Novel Signal Transduction in the Regulation of Vascular Tone and Their Involvement in Diseases

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1. Purified Rho-kinase from smooth muscle, identified as ROKα isoform, could phosphorylate both the regulatory light chain (Ser19) of smooth muscle myosin and MYPT1 subunit of myosin phosphatase (MP). Rho-kinase could be activated by arachidonic acid independently with respect to RhoA. Phosphorylation of MYPT1 at Thr695 induced the inhibition of MP activity. Using a site- and phosphorylation-specific antibody for Thr695, increased levels of MYPT1 phosphorylation at Thr695 by Rho-kinase were observed in cultured vascular smooth muscle cells by agonist stimulation.2. CPI-17, an inhibitory phosphoprotein for MP, could be phosphorylated by two RhoA downstream kinases, namely Rho-kinase and PKN. These phosphorylation converted CPI-17 to be an active inhibitory form for MP.3. The 5'-flanking region of the human MYPT1 was clone and sequenced. From this analysis MYPT1 gene was found to be a housekeeping gene.4. As MYPT1 has critical functions in MP, we knocked out the gene for MYPT1 in mice to understand the function of MP. In MYPT^<+/-> mice, the levels of MYPT1 expression in smooth muscles were similar to those in wild-type mice. No MFPT1^<-/-> newborns were obtained from the MYPT1^<+/-> intercrosses, indicating that the MYPT1 null mutation was lethal in utero. We will continue to generate the MYPT1-deficient mice using spatio-temporal control system of gene targeting.5. The changes in the expression levels of Rho-kinase-related molecules, namely RhoA, Rho-kinase, CPI-17, myosin light chain kinase, were investigated in various rat hypertensive models compared with normotensive control rats. Although these expression levels were not significantly different, the higher levels of the active form of RhoA were commonly detected in any hypertensive models. These results suggest that regardless of how hypertension begins the activation of RhoA in vascular smooth muscle is one of the principle mechanisms involved is hypertension.